Various types of substances are present in a specimen containing a target substance to be assayed, and a target substance to be assayed and a substance competitive therewith are present in a certain specimen at the same time in some cases. For example, a large portion of tartrate resistant acid phosphatase (TRACP) in serum is considered acid phosphatase derived from osteoclast and the assay of TRACP is considered useful as an indication for evaluating the function of osteoclast. Thus, TRACP is a substance that is gaining interest as a bone resorption marker (Norio Fukunaga, Toshitaka Nakamura and Toshio Matsumoto, “Osseous Metabolism Marker”, Medical Review Co., Ltd., 1995), and it has been found that besides this enzyme having enzymatic activity, fragment as degradation products of the enzyme are also present in serum (J Bone Miner Res. 15:1337-1345, 2000; and Clin Chem. 47:74-80. 2001).
By the way, acid phosphatase in serum is divided into six bands 0 to 5 from the origin by polyacrylamide gel electrophoresis. Of these, acid phosphatase corresponding to the fifth band is tartrate-resistant and is called Band 5 tartrate resistant acid phosphatase (TRACP 5: tartrate resistant acid phosphatase 5). This acid phosphatase is further divided by electrophoresis into 5a which has a high content of sialic acid bonded to a sugar chain and 5b which has almost no sialic acid bonded to a sugar chain. In addition, 5a is an enzyme derived from platelet or the like and its blood level does not vary, while only the blood level of 5b varies with bone resorption. Therefore, it is considered that 5b is the main body of tartrate resistant acid phosphatase derived from osteoclast. Also in “Clinical Chemistry” (Clin. Chem. 47:1497. 2001), it is recommended that ACP derived from osteoclast should be abbreviated as TRACP 5b. Accordingly, also in the present specification, phosphatase that refers to ACP derived from osteoclast and used as an indication of bone resorption is expressed in the term “TRACP 5b”, and tartrate resistant acid phosphatase derived from osteoclast and tartrate resistant acid phosphatase 5b (TRACP 5b ) are considered synonymous with each other. Thus, all of them are expressed in the term “TRACP 5b”.
Besides TRACP 5b, acid phosphatase derived from erythrocyte or that derived from platelet is present as ACP in a specimen. That is, when hemolysis is caused by collection of a specimen, acid phosphatase derived from erythrocyte is contained in the specimen. When serum is used as a specimen, platelets are destroyed during blood coagulation in serum preparation, so that acid phosphatase derived from platelet is contained in the specimen. Therefore, conventional methods for assaying TRACP activity do not permit assay of osteoclast-specific TRACP 5b. As a modification of these assay methods, there is known a method in which after a pretreatment comprising incubation of a 5-fold dilution of serum at 37° C. for 1hour, the residual TRACP activity is assayed by the use of p-nitrophenylphosphoric acid (pNPP) as a substrate in the presence of tartaric acid (“Nichidai-Ishi”, 49:904-911. 1990; and Clin. Chem. 33:458-462. 1987). This method permits avoidance of the influence of acid phosphatase derived from erythrocyte but does not permit exclusion of the influence of acid phosphatase derived from platelet. In addition, as a more specific activity assay method, there is a TRACP 5b assay method utilizing the difference in sensitivity to fluorine between TRACP 5b and tartrate resistant acid phosphatase derived from erythrocyte or platelet (JP-A-10-37198). This method, however, does not permit exclusion of the influence of TRACP 5a though it permits avoidance of the influence of tartrate resistant acid phosphatase derived from erythrocyte or platelet. Moreover, this method is desired to be further improved in precision because TRACP 5b activity is assayed in this method by subtracting activity not inhibited in the presence of fluorine from the total tartrate resistant acid phosphatase activity.
In immunoassay methods such as the method using a polyclonal antibody of Marius E, Sari L, et al. (J Clin Endocrinol Metab. 71:442-451. 1990; and Clin Chem. 46:1751-1754. 2000) and the method using a monoclonal antibody of Jussi M. Halleen, Heather Bull, et al. (J Bone Miner Res. 13:683-687. 1998; Immunol Lett. 70:143-149. 1999; J Bone Miner Res. 14:464-469. 1999; Clin Chem. 45:2150-2157. 1999; and Clin Chem. 46:1751-1754. 2000), activity corresponding to the whole of Band 5 is assayed, so that the influence of TRACP 5a is not negligible. In addition, the method of Halleen, et al., which is designed to assay TRACP 5b specifically, is more specific for TRACP 5b activity derived from osteoclast but indicates only slight difference between a specimen from a healthy person and a specimen from a patient suffering exacerbated bone resorption. Thus, the sensitivity of TRACP 5b as a bone resorption marker is not sufficient. The low specificity for TRACP 5b of heretofore-prepared polyclonal antibodies and monoclonal antibodies is considered as a cause for the defects described above. Since it has been reported that TRACP contains fragments with no enzymatic activity in a large amount of 10 times the amount of the active enzyme in serum, it is also conjectured that the fragments compete with the intact enzyme in serum, so that the specificity has been low because of a disadvantageous reaction system.
Even in such a case, quantification has heretofore been carried out by an immunoassay method or the like by utilizing an antigen common to the complete substance with enzymatic activity, the intact enzyme, and enzyme degradation products. However, as a result, when an epitope is limited as in the case of a monoclonal antibody, measurable fragments and non-measurable fragments appear owing to degradation. Therefore, it can be speculated that there is, for example, the following problem: even when one and the same target substance is assayed, there is no correlation among the assay results obtained by the use of various kits. In addition, it has also been reported that in the case of TRACP, examples of which have been previously given, the amount of its fragments does not clinically reflect bone resorption (J. Bone Miner. Res., 15:1337-1345, 2000; and Clin. Chem. 47:74-80, 2001). This is considered as a proper example of necessity for the measurement of only the enzyme active form. In such a case, only the enzyme level should be measured by removing the fragments. It is conjectured that in general, the assay of TRACP by an activity measurement method has been not accurate because the inactive fragments compete with the enzyme. Moreover, there has been no fragment-specific antibody for clear separation of protein fragments, so that no means for the separation has been present in itself.